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Bacterial Smear

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15 views3 pages

Bacterial Smear

Uploaded by

syedamaryamali19
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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EXPERIMENT # 6

BACTERIAL SMEAR
Object: To prepare a bacterial smear for staining.
Requirements:

 Glass slides
 Burner
 Inoculating loop
 Sterile Water
 Assorted broth and plate cultures

Theory
A bacterial smear is simply that—a small amount of culture spread in a very thin
film on the surface of the slide.
Not only are most bacteria very small, they are also very clear and difficult to view
under a microscope without first staining. You must firmly attach your bacteria to
a glass slide before you can stain them. There are two important things to consider
when preparing a slide for staining:
1. The bacteria must be evenly and lightly dispersed.
2. The bacteria need to be firmly attached to the slide so they are not washed
off during the staining procedures.
The process of making a smear preparation is an important skill in the
microbiology laboratory and is usually the first step in most staining procedures.
The quality of the smear will directly affect the quality of the subsequent staining
procedure. The smear preparation differs slightly depending on the specimen or
culture.

Smear Preparation
Smear from Plate

You should use an inoculating needle to transfer your organism to the slide. Be
sure to use sterile water to dilute your samples.

1. Label your slide. Aseptically transfer a loop-full of sterile water to the center
of the slide.

o This serves to both dilute your bacteria and give you something to
spread around.

2. Pick a well-isolated colony.

3. Prick it with your sterile needle, or slightly scoop the edge of the colony
with your sterile loop.

4. Place your needle/loop in the center of the drop and with a spiraling circular
motion spread the bacteria on the slide.

5. Set the slide aside to air dry. This will take several minutes at least. Do not
rush this step.

Fixation

The fixation procedure is the same regardless of smear source, plate or broth.
There are two methods of adhering your bacteria to the slide, heat fixation or
methanol fixation.

Heat fixing is only used with BSL1 organisms.

Methanol fixing is used with BSL2 organisms.

Heat fixing the slide can create aerosols and with BSL2 organisms, we need to
prevent this as much as possible. Methanol fixation causes fewer changes in
cellular morphology and creates no aerosols.

Please be careful when working with the methanol, if you forget you have fixed it
with methanol and your slide isn’t totally dry, the remaining methanol will catch
on fire.

Methanol Fixing
1. Be sure your slide is totally dry. Set it on the staining rack over the sink.

2. Carefully flood the slide with 95% methanol. Let it sit for two minutes.

3. Tilt the slide and pour off the methanol.

o Touch the edge of the slide to a paper towel to wick off the excess
methanol.

4. Set the slide aside to air dry before staining.

Heat Fixing

When slides are completely air-dry, heat fix the bacterial specimen by passing the
slide slowly over the flame twice.

 Heat fixing kills cells, and adheres them to the slide.


 Cells will be rinsed off the slides if they are not heat fixed properly.
 Be careful not to overheat the slides in this procedure

After heat-fixing is complete, you are ready to stain your slide.

RESULT

Bacterial smear is made, prior to staining bacteria for viewing under the
microscope.

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